Advanced optical microscopy techniques offer unique opportunities to investigate biological processes in vivo. The ability to image tissues or organisms in three dimensions (3D) and/or over time (4D imaging) permits a wide range of applications in neuroscience, immunology, cancer research, and developmental biology. (See, e.g., Mertz, Curr. Opin. Neurobiol. 14, 610-616, (2004); Kerr, J. N. D. & Denk, W., Nature Reviews Neuroscience 9, 195-205, (2008); Friedl, P., Current Opinion in Immunology 16, 389-393, (2004); Bousso, P., Current Opinion in Immunology 16, 400-405, (2004); Provenzano, P. P., et al., Trends in Cell Biology 19, 638-648, (2009); Keller, P. J., et al., Science 322, 1065-1069 (2008); McMahon, A., et al., Science 322, 1546-1550 (2008); and Mavrakis, M., et al., Development 137, 373-387, (2010), the disclosures of each of which are incorporated herein by reference.) Fundamental light-matter interactions, such as light scattering, absorption, and photo-induced biological toxicity, set the limits on the performance parameters of various imaging technologies in terms of spatial resolution, acquisition speed, and depth penetration (how deep into a sample useful information can be collected). Often, maximizing performance in any one of these parameters necessarily means degrading performance in the others. (See, e.g., Ji, N., et al., Curr. Opin. Neurobiol. 18, 605-616, (2008) and Vermot, J., et al., HFSP Journal 2, 143-155 (2008), the disclosures of each of which are incorporated herein by reference.)
Such tradeoffs in performance are seen in comparing two current well-known 4D fluorescence imaging techniques of two-photon laser scanning microscopy (2p-LSM) and one-photon light sheet (1p-LISH) microscopy: 2p-LSM excels in achieving high depth penetration in scattering tissues, while 1p-LISH allows higher acquisition speed and lower phototoxicity. In 2p-LSM, the images are generated by raster scanning the sample with tightly-focused point of near-infrared (NIR) light, inducing 2p-excited fluorescence signal only at the focus spot and thus generating 3D resolution. (See, e.g. Denk, W., et al., Science 248, 73-76 (1990) and Zipfel, W. R., et al., Nat. Biotechnol. 21, 1369-1377 (2003), the disclosures of each of which are incorporated herein by reference.) Signal and spatial resolution are maintained significantly deeper into scattering samples compared with modalities that use 1-photon excitation (such as confocal laser scanning microscopy (CLSM)), due to (i) the low scattering of NIR light, and (ii) the efficient non-imaging detection where both ballistic and scattered fluorescence photons contribute to the signal (as the 3D resolution is achieved through confinement of the excitation alone). The acquisition speed of 2p-LSM is, however, limited since the image is collected one voxel at a time.
1p-LISH microscopy is a century-old technology that has seen much development and refinement in recent years, under names ranging from Orthogonal. Plane Fluorescence Optical Sectioning (OPFOS), Thin Laser light Sheet Microscopy (TLSM), Selective Plane Illumination Microscopy (SPIM) (FIG. 1A, high-speed imaging of live zebrafish heart), Objective Coupled Planar Illumination (OCPI) (FIG. 1B, high-speed calcium imaging of neurons), ultramicroscopy (FIG. 1C, blood vessel system of mouse embryo), and Digital. Scanned Laser Light Sheet Fluorescence Microscopy (DSLM) (FIG. 1D, in toto imaging of developing zebrafish embryo), among others. (See, e.g., Siedentopf, H. & Zsigmondy, R., Ann. Phys.-Berlin 10, 1-39 (1902); Voie, A. H., et al., J. Microsc.-Oxf. 170, 229-236 (1993); Fuchs, E., et al., Opt. Express 10, 145-154 (2002); Huisken, J., et al., Science 305, 1007-1009 (2004); Holekamp, T. F., et al., Neuron 57, 661-672 (2008); Dodt, H. U. et al., Nat. Methods 4, 331-336 (2007); Huisken, J. & Stainier, D. Y. R., Development 136, 1963-1975 (2009); and Keller, P. J. & Stelzer, E. H. K., Curr. Opin. Neurobiol. 18, 624-632 (2008), the disclosures of each of which are incorporated herein by reference.)
In 1p-LISH, (FIG. 1E) a planar sheet of light is used to illuminate the sample, generating fluorescence signal over a thin optical section of the sample, which is then imaged from the direction orthogonal to the light sheet, with a wide-field imaging camera. Axial sectioning results from the thinness of the light sheet, while lateral resolution is determined by the detection optics. The orthogonal geometry between the illumination and detection pathways of 1p-LISH, compared to the collinear geometry of conventional microscopes, not only enables higher imaging speed due to the parallel image collection (millions of voxels can be imaged simultaneously), but also reduces phototoxicity since only a single focal plane of the sample is illuminated at a time. The depth penetration of 1p-LISH into scattering biological tissue, however, is limited (only slightly better than CLSM), due to (i) the imaging requirement of the wide-field detection that requires ballistic fluorescence photons only and scattered photons would degrade the image quality, and (ii) the light sheet is spatially degraded beyond its original thinness due to scattering, as it is focused deep inside an optically heterogeneous sample.
Accordingly, it would be advantageous to develop an optical microscopy technique that strikes a new balance between the imaging performance of 2p-LSM and 1p-LISH capable of providing new imaging capabilities heretofore unobtainable with conventional microscopy techniques.